Project Summary/Abstract The tectorial membrane (TM) is an extracellular matrix (ECM) structure that hovers over the organ of Corti. The TM exhibits sophisticated ultrastructure and plays important roles in frequency selection, propagation and amplification of sound waves. Malformation of the TM causes hereditary hearing deficits. Since the TM is an acellular structure, its unique properties arise from the pre-established architecture. However, the mechanism by which this exquisite structure is assembled in the extracellular space is not known. The TM is composed of both secreted proteins and glycosylphosphatidylinositol (GPI)-anchored proteins, including ?-tectorin and ?-tectorin, which crosslink TM components. While the GPI-anchorage of tectorins is conserved in vertebrates, the role of the GPI-anchor in the formation and maturation of the TM is unknown. We provide evidence that the GPI-anchorage of ?-tectorin is critical for TM development. Importantly, ?-tectorin is released from the cells by a member of the glycerophosphodiester phosphodiesterase (GDE) family, GPI-anchor cleaving enzymes. TM development is impaired in Gde3 knockout mice, indicating that both surface-tethering and release of ?-tectorin are required for normal TM development. In this proposal, we will determine the molecular mechanism by which the GPI-anchorage of ?-tectorin mediates TM formation. Importantly, we will determine how the balance between surface vs released pools of ?-tectorin shapes TM architecture. Our results will provide the first evidence of how a complex ECM structure is established and matured in the extracellular space at the molecular level, and will provide novel insights into the mechanism of hereditary and age-related hearing deficits.